Transcript Analysis of the Basal Section of Leaves Suggests
Delayed Plastid Development and Perturbation of Plastid Gene Expression
in WHY1 Knockdown Lines
In wild type leaves, transcripts associated with key developmental
processes were highly abundant in the basal section of leaves and
declined in the middle and tip sections. We therefore conducted one-way
ANOVA to determine significantly differentially abundant transcripts in
the base of wild type, W1-1 and W1-7 leaves. This identified 1267
transcripts that were differentially abundant dependent on genotype of
which 540 were not assigned a function (Table S5). The remaining 727
transcripts were assigned to a range of functions associated with
primary and secondary metabolism, RNA, DNA and protein processing,
cellular organisation and development (Table S5).
Seventy-six transcripts encoding proteins associated with plastid
biogenesis and development were differentially expressed, (Table S6; Fig
6) the majority of which were more abundant in the base of 7-day old
leaves of WHY1 knockdown plants than the wild type. These included
transcripts encoding elements of the photosynthetic electron transport
chains and Calvin cycle proteins including Rubisco (Table S6).
Furthermore, several transcripts (AK248405.1, MLOC_39198.3,
MLOC_52167.2 and MLOC_63408.1) homologous to Arabidopsis transcripts
encoding proteins required for FeS cluster assembly (Bastow, Bych,
Crack, Le Brun, & Balk, 2016; Hu, Kato, Sumida, Tanaka, & Tanaka,
2017; Roland et al., 2020; Xu, Adams, Chua, & Møller, 2005;) were less
abundant in the wild type than WHY1. Many transcripts encoded proteins
associated with the targeting and translocation of proteins to the
chloroplast as well as several proteins associated with protein folding
such as AK250352.1 and AK361117 which both exhibit homology to AT3G60370
encoding an immunophillin with protein folding activity required for the
assembly of PSII (Lima et al., 2006) and AK251420.1 encoding a
chaperonin homologous to AT2G28000 required for plastid division (Suzuki
et al., 2009). Transcripts homologous to Arabidopsis transcripts
encoding several plastid localised Clp proteases were also present
including several (MLOC_32972.1, MLOC_4257.1, MLOC_64141.1,
MLOC_68297.2, MLOC_861.2) that were previously demonstrated to play a
role in plastid biogenesis (Kim et al., 2009).
A small number of transcripts exhibited a pattern of low abundance in
W1-7. These included two transcripts (MLOC_56051.1, MLOC_56052.1)
similar to AT2G34420 encoding a chlorophyll a-b binding protein,
MLOC_61567.1 similar to plastid encoded ATCG01010 encoding an NADH
dehydrogenase subunit. Interestingly, the levels of two transcripts
(MLOC_65876.1, AK353571) with similarity to an Arabidopsis transcript
(AT2G20570; GLK1) encoding a transcription factor required for the
expression of nuclear encoded photosynthetic genes (Waters et al., 2009)
were lower in all sections of the WHY1-7 leaves.